Articles in PresS. Am J Physiol Regul Integr Comp Physiol (May 20, 2004). 10.1152/ajpregu.00100.2004
A long-term high-protein diet markedly reduces adipose tissue without major sideeffects in Wistar male rats
Magali Lacroix1, Claire Gaudichon1, Antoine Martin2, Céline Morens1, Véronique Mathé1,
Daniel Tomé1, Jean-François Huneau 1
1
UMR INRA 914, Physiologie de la Nutrition et du Comportement alimentaire, Institut
National Agronomique Paris-Grignon, 75231 Paris cedex 05, France
2
Service d’Anatomie pathologique, Hôpital Avicenne, 125 route de Stalingrad, 93000
Bobigny, France
Corresponding author: Dr Claire Gaudichon
Unité INRA 914 Physiologie de la Nutrition et Comportement Alimentaire
INA PG, 16 rue Claude Bernard, 75231 Paris Cedex 05, France
Tel. 33.1.44.08.18.29 ; fax 33.1.44.08.18.25
E-mail address: [email protected]
Key words: high protein diet, hormones, glucose, body composition, histology
Running title: Side-effects of the long-term consumption of a high protein diet
Copyright © 2004 by the American Physiological Society.
2
ABSTRACT
Whereas there is a considerable interest of high-protein, low-carbohydrate diets to manage
weight control, their safety is still the subject of considerable debate. They are suspected to be
detrimental to the renal and hepatic functions, calcium balance and insulin sensitivity.
However, the long-term effects of a high protein diet on a broad range of parameters have not
been investigated. We studied the effects of a high-protein diet in rats over a period of 6
months. 48 Wistar male rats received either a normal-protein (NP: 14 % protein) or highprotein (HP: 50 % protein) diet. Detailed body composition, plasma hormones and nutrients,
liver and kidney histopathology, hepatic markers of oxidative stress and detoxification and the
calcium balance were investigated. No major alterations of the liver and kidney were found in
HP rats, whereas NP rats exhibited massive hepatic steatosis. The calcium balance was
unchanged and detoxification markers (GSH and GST) were moderately enhanced in the HP
group. In contrast, HP rats showed a sharp reduction in white adipose tissue and lower basal
concentrations of triglycerides, glucose, leptine and insulin. Our study suggests that the longterm consumption of a HP diet in male rats has no deleterious effects and could prevent
metabolic syndrome.
3
INTRODUCTION
Despite the epidemiological evidence that protein intake increased in industrialized
countries since 3 decades (66, 25), and reached about twice the recommended intakes (72),
the question of potential adverse effects of a high protein (HP) diet remains highly
controversial. It is well established now that HP diets induce early marked metabolic changes
in man and animal models, especially when the diet contains at least 50% of energy as protein
(13, 40, 17, 42). Nevertheless, the physiological and functional consequences of a long-term
HP diet have not been fully explored.
Because of the prevalence of overweight and obesity, especially in Western countries
(41), HP diets are often considered as an alternative and successful strategy to perform
efficient and long-term weight loss (62). In fact, HP diets are currently known to induce
feeding-suppressive effects in rats (46, 5) and in humans (30, 70). Low-Calory High-Protein
diets may be successfully used for the treatment of obese subjects (3, 70). However, the
medical use of a calory restricted diet for weight-loss does not necessarily lead to a high
amount of protein intake. When dealing with spontaneous dietary intake, the contribution of
high protein intake in lowering overweight prevalence is not entirely consensual since two
epidemiological studies found a positive association between protein intake and corpulence,
either in early childhood (52, 64).
Among the suspected adverse effects of HP diets, impairment of hepatic and renal
functions are often argued, due to their central role in nitrogen metabolism. The liver and
kidney size has been shown to be enhanced after 2 wk of HP diet in rats (24, 43). Moreover,
the glomerular filtration rate has been proved to be increased by high protein intake (73, 59)
or amino acid infusion (9, 21), but the concomitant impairment of renal function in healthy
4
conditions is not demonstrated. Long-term protein intake has also been suspected to promote
insulin resistance (35, 24) and to impair cortisol secretion (60) Other adverse effects have also
been advanced such as an increase of urinary calcium excretion and a possible bone
resorption (34, 31), an enhancement of oxidative stress (47), a modification of detoxification
enzymes (49).
Given the difficulty to realize long-term interventional studies in human subjects and
the lack of complete toxicological studies on high protein diet effects, we investigated on a
wide range of biochemical, anatomical and histological parameters whether a long-term
ingestion of a HP diet could have adverse and/or beneficial effects in an obesity prone strain
of rats. For this purpose, male Wistar rats were given either a NP diet or a HP diet for 6 mo.
Detailed body composition, biomarkers of oxidative stress (reduced and oxidized glutathione,
thiobarbituric acid reactive substances, expression of J-glutamy-cysteine-ligase) and
detoxification function (Glutathione-S-Transferase), plasma hormones (insulin, cortisol and
leptine) and liver and kidney histopathology were investigated. Calcium balance was also
assessed after 4 mo of a NP or a HP diet.
MATERIALS AND METHODS
Animals and diets
All animals used in these experiments were cared for in accordance with criteria outlined in
the European Convention for the Protection of Vertebrate Animals. Experiments were carried
on Male Wistar rats (Harlan, Horst, The Netherlands) weighing 180-200 g at the beginning of
the experiment (n=48). The animals were housed in individual stainless steel wire cages in a
room with a controlled temperature (22 ± 1°C) as well as humidity and a 12 h light-dark cycle
(light 08h00 p.m.– 08h00 a.m.). Rats were fed a standard chow diet during the first 3 d
5
(Harlan Teklad TRM 9608 Rat/Mouse diet (18% protein), Harlan, Gannat, France). They
were then randomly assigned to three groups of 16 rats each. In each group, 8 rats were given
a standard normal-protein diet (NP) containing 14% total milk protein (TMP) in dry matter
(DM), and 8 rats were given a high-protein diet (HP) containing 50% TMP in DM. The
composition of the experimental diets produced by the A.P.A.E. (Atelier de Production des
Aliments Expérimentaux, INRA, Jouy-en-Josas, France) is shown in Table 1. The NP diet
was an AIN-93 modified diet: instead of casein and cysteine, this diet contained 140 g total
milk protein per kg of food. The amounts of energy, fat, cellulose, minerals and vitamins were
similar in both diets; the HP diet was lower in carbohydrate (33.6% of DM) than the NP diet
(63.6% of DM). Both diets were moistened (water/powdered diet: 1 and 2 for the NP and HP
groups, respectively) to prevent spillage. The food thus had the same consistency and
palatability in both groups. Fresh food was provided daily, at the beginning of the dark phase
(08h30 a.m.). Water levels were checked once a day and the supply renewed every two days.
During the whole experiment, the rats had free access to tap water and food. The HP and NP
groups were sacrificed 15 d, 3 mo and 6 mo after the beginning of the experimental diet
period. The animals were weighed 5 d a wk during the first two mo then twice monthly until
the end of the experiment. In order to determine food consumption, cups were weighed 5 d a
week for 1 mo, then by periods of 5 d every 3 wk until the end of the experiment.
Experimental procedure and sample collection
After 3 mo and 6 mo of ingesting either a NP diet or a HP diet, and one wk before sacrifice,
peripheral blood was taken from the cranial branch of the medial saphenous vein of overnight
fasted rats. Glycemia was immediately measured using a portable refractometer (Encore£
glucometer, Bayer Diagnostics, Puteaux, France). After blood sampling, the rats received
their regular meal.
6
After 15 d, 3 mo and 6 mo of the experimental diet, animals fasted overnight were weighed,
-1
anesthetized with sodium pentobarbital via the IP route at a dose of 30 mg.kg
(Sanofi –
Synthélabo Santé Animale, Libourne, France) and injected with 5000 IU heparin
(Laboratoires LEO, Saint-Quentin-en-Yvelines, France). The abdomen was opened and the
blood removed after rupture of both the abdominal aorta and vena cava. Blood samples were
centrifuged and the plasma was frozen at –80°C for subsequent analyses of plasma total
proteins, amino acids, insulin, leptine and cortisol. Simultaneously, the liver, kidneys and
muscles were promptly removed, washed free of gross blood with a 0.9% NaCl solution, and
weighed. The liver was sliced and immediately frozen at either –20°C
(enzyme or
biochemistry assays) or –196°C in liquid nitrogen (molecular biology assays) and then stored
at –80°C. A quarter of the liver and one of the kidneys were fixed in AFA (a 2% formalin, 5%
acetic acid, 75% ethyl alcohol solution), embedded in paraffin and stained with hematoxylineosin-saffron (HES) for subsequent histological analyses. These 3 dyes reveal the nucleus, the
cytoplasm and collagen, respectively. In addition, the Periodic Acid-Schiff’s reagent (PAS)
was used on kidneys for demonstration of basement membrane. Paraffin sections were 4 µm
thick for kidney and liver. Urine was tapped directly in the bladder and immediately frozen at
–20°C. Four deposits of white adipose tissue (WAT), epididymal, retroperitoneal, mesenteric
(including omental tissue) and subcutaneous, were removed and weighed, as well as the skin.
The other abdominal and thoracic organs were discarded and the distal parts of the limbs, the
head and the tail were severed, in order to determine the weight of this so-called "stripped"
carcass. In some of the 6 mo animals (6 rats in the NP group and 5 rats in the HP group), the
carcasses were frozen in order to determine the weight of the skeletons at a later date. Briefly,
the carcasses were boiled and the muscles and tendons carefully removed with a scalpel
blade; the remaining tissues were sifted in order to recover small bones (caudal and dorsal
7
vertebra, ribs). For all animals in the 6 mo group, the heart, adrenal glands and interscapular
brown adipose tissue (BAT) were also weighed.
An oral glucose tolerance test was performed after an overnight fast in a complementary study
performed on 12 rats (n=6 per group), after 3 mo of experiment. Fasted rats received a 1 g.kg-1
oral glucose load and blood glucose levels was measured sequentially. The results are
expressed as the difference between basal glucose and glucose at each time point.
To quantify the calcium balance, after 4 mo of the experimental diet, the 6 mo rats were
housed for 7 d in individual metabolism cages. Urine and faeces were collected daily;
crystallized thymol was added to the urine collection tubes to prevent bacterial proliferation.
Sampled urine and faeces were stored at –20°C at the end of each day of experimentation.
Analytical methods
With the exception of solvents and alcohols purchased from Carlo Erba (Val de Reuil,
France), all the chemicals used as reagents, in solutions or buffers, and all the enzymatic
assays, were obtained from Sigma-Aldrich Chimie (Saint-Quentin Fallavier, France). Urinary
and plasma urea concentrations were determined by an enzymatic assay (Urea Nitrogen
endpoint kit); albumin was revealed by its reaction with bromocresol purple (Albumin kit
BCP). GST activity was measured according to Habig's method (23). Briefly, GST conjugates
with 1-chloro-2,4-dinitro-benzene and the enzyme activity in the presence of this aromatic
substrate is determined spectrophotometrically. For oxidized (GSSG) and total (GSH)
glutathione, samples were homogenized in 9 vol. of iced NaCl 0.9%. Once filtered, 50 µl of
the solution were frozen at –20°C to perform protein quantitative analyses and 300 µl were
sampled and mixed with 100 µl of 20 % sulfosalicylic acid and centrifuged (3000g, 15 min,
4°C). The supernatant was collected and used to determine GSH and GSSG levels according
8
to the method described by Anderson (2) and Thiobarbituric Acid Reactive Substances
(TBARS) levels according to Satoh's technique (55).
Liver protein concentrations were determined using the Bicinchoninic Acid (BCA) method
(Pierce BCA-200 Protein Assay kit, Perbio Science, Bezons, France). For amino acid and
plasma protein analysis, 1 ml of plasma was deproteinized with 40 µl of 100 % sulfosalicylic
acid, stored at 4°C for 1 h, then centrifuged (3000 g, 4°C, 15 min). The supernatant was dried
and resuspended in a lithium citrate buffer (pH 2.2) for analysis. The plasma protein pellet
was lyophilized. Total nitrogen in plasma proteins was measured with an elemental nitrogen
analyzer (EA5NA 1500 II, Fisons Instruments, Manchester, UK) with atropine (Carlo Erba
Instruments, Arceuil, France) as a standard, as previously described (18). Protein
concentrations were calculated using a conversion factor of 6.25, originating from the
standard amount of nitrogen in protein, i.e. 16%. Plasma amino acid concentrations were
determined using an HPLC system (Bio-Tek Instruments, St Quentin en Yvelines, France)
combined with post-column ninhydrin derivatization. Separation was performed on a cation
exchange resin (Biorad Dowex AG-50X8, Interchim, Montluçon, France). All amino acids
were detected at 540 nm, except for proline (440 nm). Gamma-amino-butyric acid was used
as an internal standard. Plasma creatinine, triglycerides, total and HDL-cholesterol assays
were performed using a Mascott + 3X spectrophotometer (Lisabio, Pouilly-en-Aixois,
France).
Calcium levels in the urine and faeces (as well as in drinking water and food) were
determined using a flame atomic absorption spectrometer (model SpectrAA 50, Varian, Les
Ulis, France) with an air/acetylene burner (reducing flame). Before analysis, liquid samples
-1
were diluted 1/100 in a spiking solution containing 5 g.l of lanthanum chloride (La2O3), 1
-1
-1
g.l of calcium chloride and 0.2 g.l of potassium chloride. Solid samples (faeces and diets)
9
were mineralized by heating for 2 h (100°C) in 25 vol of 2N nitric acid. The wavelength of
the cathode lamp was 422.7 nm.
To measure changes in J-GCL (J-glutamy-cysteine-ligase) mRNA expression levels after 3
mo of the experimental diet, we chose a semi-quantitative RT-PCR protocol. RNA extraction
was carried out on liver samples from 4 animals in each group using the RNeasy“ Mini Kit
(Quiagen, Courtaboeuf, France), according to the manufacturer’s instructions. The following
primers (Genset Oligos, La Jolla (CA), USA) were used to amplify J-GCL mRNA using a
one-step RT-PCR kit (Quiagen, Courtaboeuf, France) and
33
P-dATP (PerkinElmer Life
Science and Products, Courtaboeuf, France): 5’CAAGAACACACCATCTCCGTTTG-3’
(forward) and 5’ ACTGTACCTCCATTGGTCGGAAC-3’ (reverse). Running PCR without
prior reverse transcription controlled the absence of DNA contamination. Amplification was
performed in a PTC-200 thermocycler (MJ Research, Watertown (MA), USA) for 24 cycles.
To enable semi-quantitative analysis, E-actin mRNA were co-amplified using the following
primers:
5’-TGGAATCCTGTGGCATCCATGAAA-3’
(forward)
and
5’-TAAAACGCAGCTCAGT AACAGTCCG-3’ (reverse). The RT-PCR products were then
separated by electrophoresis on a 2 % agarose gel stained with ethidium bromide, and
quantified using a digital imaging system (Alpha Innotech Corporation, San Leandro, USA).
Concurrently, quantification was also performed by measuring the amount of
33
P-dATP
recovered in the amplification products using liquid scintillation counting. Briefly, BETstained spots were punched, solubilized in chaotropic buffer (QX1, Quiagen, Courtaboeuf,
France) and 3 ml of a liquid scintillation cocktail (Ultima Gold¥, PerkinElmer Life Science
and Products, Courtaboeuf, France) was added. The radioactivity was determined using a
Packard TriCarb 2200 CA liquid scintillation analyzer (PerkinElmer, Courtaboeuf, France).
For each quantification method, the results were expressed as the ratio of amplification
products in the mRNA of interest to those of the housekeeping mRNA.
10
125
Plasma hormones were quantified using radioimmunoassay kits with [
I] as a tracer (INSIK-
5 and GammaCoat• [125I] Cortisol RIA, (Diasorin, Antony, France) for insulin and cortisol,
and LEP-R61 (Mediagnost, Tuebingen, Germany) for leptine). The radioactivity of the tracer
was quantified in a gamma scintillation counter (Cobra II Auto-Gamma, Perkin Elmer,
Courtaboeuf, France).
Plasma homocysteine concentrations were determined using a competitive enzyme
immunoassay with peroxidase as the enzymatic system Axis Homocysteine (EIA).
Kidney and liver specimens were processed in the Histopathology Department of the
Avicenne Hospital (Bobigny, France). Tissues were dehydrated and immersed in liquid
paraffin. Thin sections (0.1 µm) were pasted on slides and stained with HES (Hematoxylin
Eosin Saffron). Histological sections were examined under a Leica DM LB microscope (Leica
Microsystems, Rueil-Malmaison, France).
Statistics
The results were expressed as means with their standard errors (± SEM). The effects of diet
and age were tested by two-way analysis of variance, using the GLM procedure of SAS (SAS
version 6.11, Cary, NC, USA). An alpha-level of 0.05 was set to determine significance.
11
RESULTS
Energy intake, body weight, body composition and calcium balance :
Whatever the diet, food intake varied from one day to another, but no refusal or unexpected
reduction in food intake was observed during the 6 mo period. The mean daily food intake
after 6 mo of experiment differed significantly between HP rats (348.8 ± 3.1 kJ) and NP rats
(388.2 ± 6.8 kJ). The energy intake in HP rats was 87% of that seen in NP rats. After 6 mo of
the experimental diet, a significant reduction in body weight was observed in HP rats
compared to NP rats. The weight of HP rats was 82% of that seen in NP rats.
Body weight, white adipose tissue (WAT) and stripped carcass weights increased
significantly with age (p< 0.05). After 15 d of an experimental diet (results not shown), body
compositions exhibited no differences between the groups except regarding the kidneys (1.99
± 0.11 and 2.29 ± 0.25 g for NP and HP groups, respectively, p<0.05). However, after 6 mo of
the experimental diet, the body composition showed marked differences in WAT, since this
represented 27.5% of body weight in NP rats but only 19.3 % in HP rats (Table 2).
Subcutaneous WAT in NP rats was double that in HP rats, whereas the respective stripped
carcass weights were similar in NP and HP rats. Therefore, WAT was the main contributor to
the increased body weight of NP rats when compared to HP rats. The BAT weight was twice
as high in NP rats as in HP rats. No differences between groups were observed with respect to
skeleton weight after 6 mo of adaptation.
Quantifications of calcium intake (food and water) after 4 mo (2549 ± 70 and 2900 ± 64
mg/animal/wk in NP and HP rats, respectively; p<0.05), calcium excretion in faeces (887 ± 51
and 1413 ± 91 mg/animal/wk in NP and HP rats, respectively; p<0.05) and in the urine (22 ±
2 and 38 ± 5 mg/animal/wk in NP and HP rats, respectively; p<0.05) led to a positive balance
in both groups, with no significant difference between NP and HP rats (1640 ± 79 and 1448 ±
99 mg/animal/wk in NP and HP rats, respectively; NS).
12
Plasma hormones, glucose, proteins, amino acids and lipids
Fasting plasma glucose, insulin, leptine and cortisol concentrations were significantly higher
in NP rats than in HP rats (Table 3). There was also a significant effect of age on blood
leptine, insulin and sugar levels, with a dramatic increase in leptine with age (p<0.0001).
Moreover, glucose tolerance tended to be improved in HP rats (Figure 1). The areas under the
curve were 103.8 ± 12.3 'mmol x min in the HP group and 138.3 ± 14.1 'mmol x min in the
NP group. However, these values did not significantly differ (p=0.09).
Plasma urea was systematically higher in HP rats than in NP rats (p < 0.01), whatever the
duration of the experiment (4.5 ± 0.2 vs. 3.4 ± 0.2 mmol/l for HP and NP rats after 6 mo of
experiment) (Table 3). Plasma creatinine tended to be higher in NP rats but did not reach the
significance when all groups (15 d, 3 and 6 mo) were taken in account (p=0.053). A
significant difference was observed in rats slaughtered after 3 mo. Plasma homocysteine
levels remained unchanged throughout the experiment. Plasma albumin, when expressed
either as an absolute value or as a percentage of total plasma proteins, and plasma protein,
varied with age (p < 0.01) but the diet had no effect. Circulating amino acid values showed
few differences dependent upon age or diet. Indispensable amino acids decreased significantly
with age. Leucine and isoleucine concentrations were significantly higher in the HP group
whereas threonine concentrations were lower (not shown).
Plasma cholesterol and triglycerides varied significantly with age (Table 3). Furthermore,
triglyceride levels were significantly higher in NP rats (p=0.0009) whereas diet had no effect
on total and HDL cholesterol.
13
Oxidative stress-related parameters and histological study
Hepatic oxidative stress was quantified by measuring TBARS and glutathione levels (Table 4).
Oxidized glutathione concentrations were negligible. Total liver glutathione levels rose with
age in HP rats but not in NP rats (age x regime effect, p<0.004). TBARS liver concentrations
did not differ in HP rats when compared with NP animals. GST activity markedly increased
with age (p<0.0001) and was higher in HP rats (p=0.0006).
RT-PCR products are visualized on ethidium bromide stained agarose gel (Figure 2). The
quantification of J-GCL mRNA expression after 3 mo of experiment did not differ between
groups, whatever the method (ratio: 0.10 ± 0.03 vs. 0.09 ± 0.01 in HP and NP rats respectively
in the radioactivity assay, and 0.10 ± 0.00 vs. 0.11 ± 0.00 in HP and NP rats respectively in the
agarose gel assay).
No serious histological lesions were observed in either group after 3 or 6 mo of experiment.
Figure 3 shows typical histological sections of liver (HES stain). All NP rats systematically
exhibited marked hepatic micro- and macro-vacuolar steatosis, which was probably related to
their excessive adiposity (Figure 3A1 and 3A2). In contrast, no steatosis was found in HP rats
(Figure 3B). Minor, non-pathological inflammatory lesions were found in the liver of 2 rats in
the HP group after 6 mo of the experimental diet (not shown). Figure 4 shows histological
kidney glomeruli sections (PAS stain). Despite the relative thickness of the sections (4µm),
focus adjustment permitted careful examination of renal tissues. The capillary loops of the
glomeruli were well defined and thin. No nephrocalcinosis, no area of collagenous sclerosis or
hypercellular glomerulus was detectable. Endothelial and mesangial cells were normal and so
were the surroundings tubules. Similarly to PAS stain, HES stain of kidneys (not shown) did
not reveal any noticeable lesion. However, one rat of the HP group exhibited a localized and
limited interstitial lymphocytic inflammatory nodule and another HP rat showed a minor
kidney lesion indicative of moderate inflammation of the renal papilla (not shown).
14
DISCUSSION
The aim of this study was to estimate the consequences of a long term intake of high levels of
protein in an obesity-prone rat strain. The results showed that the long-term ad libitum
consumption of a high-protein diet markedly reduced food intake and lowered white adipose
tissue, in accordance with several mid-term studies. Concomitantly, basal blood insulin,
leptine and triglyceride levels, and glucose tolerance was improved. Calcium balance was
unaffected by a high intake of milk proteins. Moreover, in contrary to what has generally
been admitted, no adverse effects of the HP diet were reported, particularly regarding hepatic
and renal histopathology.
After 6 mo of the experiment, the body weight of rats fed the HP diet was 18% lower than
that of rats fed the NP diet. Body composition measurements revealed remarkable differences
between the two groups, especially concerning the subcutaneous fat pad. These results are in
agreement with those obtained during short-term studies (3 wk) (39, 29, 42) and can be
explained to some extent by the reduction in caloric intake of 13%. This decrease in energy
consumption was most probably due to the greater sensory-specific satiety of protein
compared to carbohydrate (65, 37, 5). The ratio of energy intake to body mass was higher in
NP rats (0.95 kJ/g) than in HP rats (0.68 kJ/g) whereas the ratio of energy intake to lean body
mass is similar in both groups (1.72 kJ/g in NP rats vs. 1.69 kJ/g in HP rats). In consequence,
the surplus of energy intake in NP rats has probably been used to generate fat tissues.
However, the subsequent reduction of 58 % in WAT could also arise from an increased
thermogenic response to a protein meal intake (61). HP diets are good candidates for weight
reduction, especially in obese female subjects (44). A 6 mo randomized trial concerning a
protein vs. carbohydrate fat-reduced diet showed that with the HP diet, the proportion of
obese subjects achieving clinically relevant weight loss was greater than with a NP diet (58).
15
However, some studies have reported no specific effect of HP diets on reducing body weight
or body fat in obese subjects (3, 48).
In the present study, the weight reduction in rats fed the HP diet was strongly associated with
lower basal blood sugar and insulin levels, as previously described (68) and improved glucose
tolerance. As the isocaloric exchange between HP and NP diets was performed on
carbohydrate, the flattening effect of the HP diet on insulin and glucose basal levels could be
attributed to its reduced carbohydrate content. This contrasts with the results of many studies
that have acknowledged the fact that high protein intakes induce an increase in glucose and
insulin concentrations (7, 54, 35). The oxidative conversion of amino acids to glucose
resulting from HP feeding has been postulated to exert a negative metabolic effect on the
glycolysis / gluconeogenesis balance in the liver, through a modification of the activity of key
enzymes involved in glucose metabolism (8, 45). This assumption forms the scientific basis
for the theory of amino acid-glucose competition as a factor in insulin resistance. However,
many of the previous data do not fully support this theory as they were obtained either with a
simultaneous load of glucose and amino acids, or without strictly controlled diets. In contrast,
the results of a randomized controlled trial on obese glucose-tolerant subjects showed that a
low calorie HP diet could improve insulin sensitivity after 21 d of experiment (48) and
achieve a normalization of insulin levels within 4 wk (3). Although the present study does not
explore insulin sensitivity, it shows that the long-term isocaloric exchange between protein
and carbohydrate is favorable regarding the basal concentrations of insulin and glucose and
tends to improve glucose tolerance. This is in accordance with the fact that weight loss is
often associated with improved glucose tolerance and insulin sensitivity related to enhanced
glucose oxidation (15, 16, 20). Moreover, rats fed a HP diet seemed to develop less leptine
16
resistance with age than rats fed a NP diet, which is in line with their marked reduction of
adipose tissue (27, 12).
Many studies in obese and overweight hyperinsulinemic humans have shown a decrease in
basal plasma cholesterol levels associated with weight loss, irrespective of the type of diet
(44, 10). Although a hypercholesterolemic effect of casein has often been suspected,
especially when compared with a legume protein source (38, 22), we did not find any increase
in circulating cholesterol in HP rats consuming three times more milk protein than NP rats. In
agreement with studies performed in humans (14, 33), we also reported a stabilization of
serum triacylglycerol concentrations with age in HP rats, whereas triglycerides increased from
15 d to 6 mo in NP rats. This difference was probably related to the lower carbohydrate
content of the HP diet.
Given the roles of the liver and kidneys in amino acid and nitrogen metabolism, HP diets have
been suspected of having potentially deleterious effects on the structure and function of these
organs. The liver was identified early as a major actor in urea and glutamine metabolism (32).
The rate of urea production varies linearly with protein intake in healthy adults (74). The
activity of several enzymes, such as threonine-dehydratase, transaminases and glutaminase, is
known to be dramatically increased in animals fed HP diets (56, 69, 46, 51, 29). The increase
in mitochondrial oxidation in the liver cells of rats fed HP diets (51, 26) has led to the
conclusion that oxidative stress was enhanced with this diet. Accordingly, we found a
moderate but significant elevation of oxidative activities, as reflected by hepatic GSH levels
and GST activities. However, TBARS and the expression of J-GCL were unaffected by the
diet, and GSH value in the HP group remained in a physiological range. Our results show that
the effect of a high-protein intake on oxidative stress is controversial, as can be illustrated by
17
the results of Petzke et al. (47), who found no alteration in blood or hepatic GSH levels after
the 4-mo ad libitum ingestion of a 51.3% protein diet.
In addition, histopathological examination of liver slices did not reveal any pathological
lesions in periportal or perivenous hepatocytes. To our knowledge, histopathology has been
seldom, if ever, performed on animals fed HP diets in the long term and these are important
findings. In the same way, the glomerular hyperfiltration induced by elimination of the end
products of AA metabolism has led to the conclusion that HP diets could be detrimental to the
renal function. Basic AA loads have been shown to increase water consumption in rats (1),
and indeed, we also found a 1.4-fold rise in water consumption in rats receiving the HP diet
(data not shown). A higher water consumption is likely to increase glomerular filtration rate
and this could explain the trend of a lower plasma creatinine that we observed in HP rats. To
our knowledge, the only study that ever documented a toxic effect of a HP diet is that of
Stonard (63) who reported that a moderate HP diet (33 %) was associated with
nephrocalcinosis and tubular damage in a specific strain of female rats (Alderley Park).
However, a significant occurrence of renal damage was observed in rats fed single-cell
proteins but not in rats fed casein, suggesting a specific effect of this protein source. A
moderate restriction of dietary protein has also been demonstrated to slow the progression of
renal impairment in rats (6, 11, 50) and in human patients (53, 75, 19), but did not provided
any benefit in healthy individuals (67). On the other hand, a prospective trial on the effects of
protein restriction on the progression of chronic renal insufficiency in 456 adult patients
showed no correlation between a worsening of renal failure and the protein catabolic rate (36).
Nevertheless, none of these findings contradicts the absence of pathological findings in our
study when we explored the potential adverse effects of HP diets on the renal function of
healthy male animals. Although these results are hardly transposable to humans, especially
because the capacity to concentrate urea is several fold higher in rodents than in humans, it is
18
interesting to note that a recent study showed that dietary intervention for 6 mo in 65 obese
subjects consuming a 25% protein diet caused adaptative changes in renal size and GFR
without any indications of adverse effects (59). To further conclude to the absence of renal
toxicity of HP diets, a longer-term study should be necessary as well as a study on rat females
in which lesions can be produced more readily, as mentioned in the work of Stonard et al.
(63).
Excess dietary protein has also been suspected of generating large quantities of acids
(phosphates and sulfates), excreted by the kidneys to maintain pH homeostasis. This increase
in net acid excretion is coupled with calciuria, the skeleton thus supplying a buffer by bone
resorption (71). Some authors have postulated that HP diets cause a pathological impairment
of calcium homeostasis, because the long-term consequences of even a small change in the
calcium balance are substantial (4, 28). However, there is no clear evidence that HP diets
alone cause osteopenia. Recently, Skov et al. (57) found that a 6-mo HP intake was associated
with a slowing in bone mineral loss in obese subjects. Calcium excretion and markers of bone
turnover were reported to be unchanged in overweight subjects consuming a 15 or a 27 %
protein diet for 16 wk (14). In our study, the increased calcium losses observed in the HP
group were balanced by higher intakes, due to the association of calcium and caseinates. In
consequence, no alteration to the calcium balance was observed, in accordance with the
similar skeleton weights observed in both groups. However, it must be pointed out that
calcium intake in the HP group was important due to the use of milk protein as the sole
protein source, and calcium balance may have been different using another protein source.
19
CONCLUSION
We performed this study in order to explore whether or not HP diets might be detrimental to
healthy animals over a long period representing a quarter of their life span. It would be almost
impossible to perform a study on an equivalent period in humans. We can conclude that in
male rats, a protein intake of three times the requirements did not produce any adverse effects
on the renal and hepatic functions, on oxidative stress or on the calcium balance. On the
contrary, exchanging carbohydrates for proteins was beneficial regarding body composition,
basal triglycerides, glucose, leptine and insulin plasma concentrations. The results of the
present study agree with the idea that long-term dietary management is of major importance
to preventing obesity and its correlative X syndrome. Further research is required to specify
the respective effects of weight loss and high protein intake on glucose and lipid metabolism.
20
Acknowledgments
We are very grateful to Cecile Bos, Ahmed Bensaïd, Jean-Christophe Bouthegourd and
Catherine Luengo for their skilful help with slaughtering the animals, Christiane LarueAchagiotis for her assistance with leptine assays, and Alain Bermond from the Analytical
Chemistry Unit of the INA-PG for assistance with calcium measurements.
21
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29
Table 1 Composition of diets
Normal-protein
High-protein
(14 % protein, P14)
(50% protein, P50)
Total milk protein (TMP)
140
500
Pregelatinized cornstarchA
569.9
312.7
SucroseB
92.8
50
Soybean oilD
40
40
Mineral mixtureE (AIN93-M)
35
35
Vitamin mixtureE (AIN93-V)
10
10
Alpha-celluloseF
50
50
CholineE
2.3
2.3
TOTAL
1000
1000
Energy (kJ/g)
15.9
15.8
Nutrient (g/kg)
A
Cerestar, Haubourdin, France
B
Eurosucre, Paris, France
C
APAE-INRA, Jouy-en-Josas, France
D
ICN Biochemicals, Orsay, France
E
ICN Biomedicals, Costa Mesa, California
F
Medias Filtrants Durieux, ZI Torcy, France
30
Table 2 Body weight and body composition of rats after 3 mo and 6 mo ad libitum
consumption of either a normal-protein (NP, 14% of protein) or high-protein diet (HP, 50% of
protein).
3 mo
6 mo
NP
HP
NP
HP
519.6 ± 22.9
465.50 ± 12.0
620.1 ± 34.4
510.63 ± 15.3
Liver (g) AB C
12.6 ± 0.5
12.0 ± 0.3
14.3 ± 0.7
11.8 ± 0.3
Kidney (g) AB
2.5 ± 0.1
2.8 ± 0.1
2.6 ± 0.1
2.7 ± 0.1
Spleen (g)
0.7 ± 0.0
0.8 ± 0.0
0.8 ± 0.0
0.8 ± 0.1
11.2 ± 0.4
11.1 ± 0.3
10.6 ± 0.4
10.7 ± 0.3
105.6 ± 11.9
64.3 ± 2.1
170.6 ± 20.1
98.8 ± 12.3
20.0 ± 1.5
13.8 ± 0.3
27.0 ± 1.7
19.0 ± 2.0
- epididymal (g) AB
20.2 ± 1.9
13.5 ± 0.6
27.4 ± 2.8
18.3 ± 2.5
-retroperitoneal (g) AB C
23.6 ± 2.0
15.6 ± 0.9
35.1 ± 3.6
20.8 ± 2.5
-subcutaneous (g) AB C
41.4 ± 6.4
23.6 ± 1.2
80.3 ± 12.0
42.7 ± 6.1
-mesenteric (g) AB C
20.3 ± 3.6
11.5 ± 0.6
27.7 ± 2.1
17.0 ± 1.9
ND
ND
4.4 ± 0.3
2.7 ± 0.3
80.4 ± 3.7
69.8 ± 1.7
86.5 ± 3.2
83.1 ± 4.7
15.5 ± 0.2
15.0 ± 0.4
14.0 ± 0.3
16.2 ± 0.7
200.9 ± 7.1
195.9 ± 6.9
224.7 ± 9.1
206.5 ± 5.8
38.9 ± 1.2
42.0 ± 0.6
36.4 ± 0.7
40.7 ± 1.5
ND
ND
11.56 ± 1.25
11.70 ± 0.90
Body weight (g) AB C
Gut (g)
A
White adipose tissue (g) AB C
Expressed as % of BW AB
Brown adipose tissue (g) B
Skin (g)
AB
Expressed as % of BW AC
“Stripped carcass” (g) A
Expressed as % of BW AB
Skeletons (g)
A
Significant effect of age (ANOVA, p<0.05)
B
Significant effect of diet (ANOVA, p<0.05)
C
Significant effect of age x diet (ANOVA, p<0.05)
Stripped carcass: cleaned out carcass – skin – tail – head - distal part of the limbs
31
Table 3 Fasting plasma levels of hormones, glucose, proteins, amino acids, homocysteine,
urea, creatinine and lipids in rats after 15 d, 3 mo and 6 mo ad libitum consumption of either a
normal-protein (NP, 14% of protein) or high-protein diet (HP, 50% of protein).
15 d
NP
3 mo
HP
NP
6 mo
HP
NP
HP
Insulin (mIU/l)AB
75.2 ± 2.4 62.6 ± 8.0 75.8 ± 5.9
66.5 ± 4.1
122.8 ± 21.4 90.3 ± 3.5
Cortisol (µg/l)B
21.0 ± 1.6 14.4 ± 0.1 22.3 ± 3.1
15.0 ± 1.7
23.8 ± 4.5
14.2 ± 1.0
9.0 ± 2.2
34.2 ± 4.9
17.2 ± 1.1
46.9 ± 4.0
26.4 ± 3.7
5.33 ± 0.17
4.44 ± 0.17
4.99 ± 0.17
4.11 ± 0.11
3.5 ± 0.2
6.6 ± 1.0
3.4 ± 0.2
4.5 ± 0.2
38.3 ± 3.2
54.8 ± 7.6
52.8 ± 5.8
-1
Leptine (µg/l )AB
Glycemia (mmol/l)AB
ND
6.8 ± 1.3
ND
Urea (mmol/l)B
3.8 ± 0.5
Creatinine (µmol/l)AB
35.4 ± 5.3 32.0 ± 4.0 47.0 ± 6.7
5.0 ± 0.4
Protein (g/l)
Total proteinA
61.0 ± 1.3 58.0 ± 1.8
65.4 ± 0.9
63.6 ± 2.9 55.2 ± 1.9
56.4 ± 1.1
AlbuminA
42.6 ± 1.9 41.3 ± 1.6
42.2 ± 2.2
43.6 ± 1.1 49.1 ± 1.3
46.5 ± 1.2
892 ± 47
828 ± 36
Amino acids (µmol/l)
IndispensableA
1166 ±74
1202 ± 162
959 ± 52
Dispensable
2217 ± 105
2260 ± 317
2070 ± 106† 1776 ± 85 2049 ± 144
2090 ± 84
Neoglucogenic
1628 ± 66
1989 ± 271
1953 ± 93
1548 ± 63 1335 ± 93
1913 ± 83
Homocysteine
6.9 ± 1.0
6.6 ± 1.2
8.7 ± 1.3
8.8 ± 1.1
8.8 ± 0.9
926 ± 53
6.5 ± 1.0
Lipids (mmol/l)
TriglyceridesAB
0.76 ± 0.09 0.56 ± 0.08 0.86 ± 0.10 0.66 ± 0.10 1.28 ± 0.21
0.68 ± 0.07
Total cholesterolA
1.79 ± 0.13 1.53 ± 0.13 1.54 ± 0.06 1.53 ± 0.17 2.14 ± 0.18
1.89 ± 0.12
HDL-cholesterolA
0.90 ± 0.07 0.77 ± 0.08 0.53 ± 0.03 0.58 ± 0.10 0.76 ± 0.06
0.77 ± 0.04
A
Significant effect of age (ANOVA, p<0.05)
B
Significant effect of diet (ANOVA, p<0.05)
32
Table 4 Hepatic levels of Total glutathione (GSH), oxidized glutathione (GSSH) TBARS
and Glutathione-S-Transferase (GST) activity after 15 d, 3 mo and 6 mo ad libitum
consumption of either a normal-protein (NP, 14% of protein) or high-protein diet (HP, 50% of
protein).
15 d
3 mo
6 mo
NP
HP
NP
HP
NP
HP
GSH (µg/mg protein)C
16.3±1.3
17.7±0.9
16.6±0.2
19.8±0.7
21.3±0.8
16.8 ± 0.2
GSSG (µg/mg protein)
0.10±0.05
0.10±0.10
0.08±0.06
0.20±0.20
0.50 ± 0.30
0.20 ± 0.20
TBARS (µg/mg protein)
2.1±0.2
1.7±0.1
2.3±0.2
2.0±0.2
1.6 ± 0.3
2.1 ± 0.4
GST (mIU/mg protein)AB
67.5±5.7
88.9±16.3
98.9±2.6
118.4±2.2
104.5±4.4
128.3±4.1
A
Significant effect of age (ANOVA, p<0.05)
B
Significant effect of diet (ANOVA, p<0.05)
C
Significant effect of age x diet (ANOVA, p<0.05)
33
-1
Glucose tolerance test ( Delta mmol.l )
4
HP
NP
Areas under the curves (mmolxmin)
160
140
D glycemia (mmol.l-1)
3
120
100
80
60
40
2
20
0
HP
NP
1
0
0
20
40
60
80
100
120
140
time (min)
Figure 1 : Oral glucose tolerance tests in rats after 3 mo ad libitum consumption of either a
normal protein (NP, 14% of protein) or high protein diet (HP, 50% of protein).
34
Triplicates of
liver samples
(HP diet)
Standard 100bp
DNA ladder
Triplicates of
liver samples
(NP diet)
Row A
Row B
Figure 2 : RT-PCR analysis of J-GCL in liver cells of rats after 3 mo of ad libitum
consumption of either a normal protein (NP, 14% of protein) or high protein diet (HP, 50% of
protein). This RT-PCR was developed using specific oligonucleotides and
33
P-dATP.
Deposits corresponded to triplicates from different rats submitted to either a NP or HP diet
for 3 mo. The reaction products were analyzed by agarose gel electrophoresis followed by
ethidium bromide staining. Row A shows the results of RT-PCR in rat liver cells for the
amplified fragment of J-GCL (668 pb). The expected PCR product for E-actin (353 bp) in rat
liver cells was observed on row B.
35
microvacuolar
steatosis
macrovacuolar
steatosis
A1
A2
B
Fig. 3 : Liver histology sections in rats after 6 mo ad libitum consumption of either a normal
protein (NP, 14% of protein) or high protein diet (HP, 50% of protein). A1: Liver section of a
rat after 6 mo consumption of a NP diet; this picture shows the microvacuolar and
macrovacuolar steatosis that was observed in the 8 rats in this
group. A2: Detail of
macrovacuolar steatosis (note the nucleus pushed aside of the cell membrane). B: Liver
section of one rat after 6 mo ingestion of a HP diet.
36
A
B
Fig. 4 : Kidney histology sections in rats after 6 mo ad libitum consumption of either (A) a
normal protein (NP, 14% of protein) or (B) high protein diet (HP, 50% of protein).